text: Several methods have been developed that make use of the estimated (via 1H or 13C shifts) or predicted (via sequence) secondary structure content of the protein being analyzed. These programs include PSSI, CheckShift, LACS, and PANAV.
  The PSSI and PANAV programs use the secondary structure determined by 1H shifts (which are almost never mis-referenced) to adjust the target protein’s 13C and 15N shifts to match the 1H-derived secondary structure. LACS uses the difference between secondary 13Cα and 13Cβ shifts plotted against secondary 13Cα shifts or secondary 13Cβ shifts to determine reference offsets. A more recent version of LACS [68] has been adapted to identify 15N chemical shift mis-referencing. This new version of LACS exploits the well-known relationship between 15N shifts and the 13Cα (or 13Cβ shifts of the preceding residue. In contrast to LACS and PANAV/PSSI, CheckShift uses secondary structure predicted from high-performance secondary structure prediction programs such as PSIPRED  to iteratively adjust 13C and 15N chemical shifts so that their secondary shifts match the predicted secondary structure. These programs have all been shown to accurately identify mis-referenced and properly re-reference protein chemical shifts deposited in the BMRB,. Note that both LACS and CheckShift are programmed to always predict the same offset for 13Cα and 13Cβ shifts, whereas PSSI and PANAV do not make this assumption. As a general rule, PANAV and PSSI typically exhibit a smaller spread (or standard deviation) in calculated reference offsets, indicating that these programs are slightly more precise than either LACS or CheckShift. Neither LACS nor CheckShift are able to handle proteins that have the extremely large (above 40 ppm) reference offsets, whereas PANAV and PSSI seem to be able to deal with these kinds of anomalous proteins.
  In a recent study, a chemical shift re-referencing program (PANAV) was run on a total of 2421 BMRB entries that had a sufficient proportion of (>80%) of assigned chemical shifts to perform a robust chemical shift reference correction. A total of 243 entries were found with 13Cα shifts offset by more than 1.0 ppm, 238 entries with 13Cβ shifts offset of more than 1.0 ppm, 200 entries with 13C’ shifts offset of more than 1.0 ppm and 137 entries with 15N shifts offset by more than 1.5 ppm. From this study, 19.7% of the entries in the BMRB appear to be mis-referenced. Evidently, chemical shift referencing continues to be a significant, and as yet unresolved problem for the biomolecular NMR community.